1. Field of the Invention
The present invention relates to thermostable DNA polymerases which have been altered or mutated such that a different level of 5' to 3' exonuclease activity is exhibited from that which is exhibited by the native enzyme. The present invention also relates to means for isolating and producing such altered polymerases. Thermostable DNA polymerases are useful in many recombinant DNA techniques, especially nucleic acid amplification by the polymerase chain reaction (PCR) self-sustained sequence replication (3SR), and high temperature DNA sequencing.
2. Background Art
Extensive research has been conducted on the isolation of DNA polymerases from mesophilic microorganisms such as E. coli. See, for example, Bessman et al., 1957, J. Biol. Chem. 223:171-177 and Buttin and Kornberg, 1966, J. Biol. Chem. 241:5419-5427.
Somewhat less investigation has been made on the isolation and purification of DNA polymerases from thermophiles such as Thermus aquaticus, Thermus thermophilus, Thermotoga maritima, Thermus species sps 17, Thermus species Z05 and Thermosipho africanus. The use of thermostable enzymes to amplify existing nucleic acid sequences in amounts that are large compared to the amount initially present was described in U.S. Pat. Nos. 4,683,195 and 4,683,202, which describe the PCR process, both disclosures of which are incorporated herein by reference. Primers, template, nucleoside triphosphates, the appropriate buffer and reaction conditions, and polymerase are used in the PCR process, which involves denaturation of target DNA, hybridization of primers, and synthesis of complementary strands. The extension product of each primer becomes a template for the production of the desired nucleic acid sequence. The two patents disclose that, if the polymerase employed is a thermostable enzyme, then polymerase need not be added after every denaturation step, because heat will not destroy the polymerase activity.
U.S. Pat. No. 4,889,818, European Patent Publication No. 258,017 and PCT Publication No. 89/06691, the disclosures of which are incorporated herein by reference, all describe the isolation and recombinant expression of an .about.94 kDa thermostable DNA polymerase from Thermus aquaticus and the use of that polymerase in PCR. Although T. aquaticus DNA polymerase is especially preferred for use in PCR and other recombinant DNA techniques, there remains a need for other thermostable polymerases.